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Abstract Background The CRISPR-Cas13 system is an RNA-guided RNA-targeting system and has been widely used in transcriptome engineering with potentially important clinical applications. However, it is still controversial whether Cas13 exhibits collateral activity in mammalian cells. Results Here, we find that knocking down gene expression using RfxCas13d in the adult brain neurons caused death of mice, which may result from the collateral activity of RfxCas13d rather than the loss of target gene function or off-target effects. Mechanistically, we show that RfxCas13d exhibits collateral activity in mammalian cells, which is positively correlated with the abundance of target RNA. The collateral activity of RfxCas13d could cleave 28s rRNA into two fragments, leading to translation attenuation and activation of the ZAKα-JNK/p38-immediate early gene pathway. Conclusions These findings provide new mechanistic insights into the collateral activity of RfxCas13d in mammalian cells and warn that the biosafety of the CRISPR-Cas13 system needs further evaluation before application to clinical treatments.

Abstract The innate immune system is the first line of host defense, which responds rapidly to viral infection. Innate recognition of viruses is mediated by a set of pattern recognition receptors (PRRs) that sense viral genomic nucleic acids and/or replication intermediates. PRRs are mainly localized either to the endosomes, the plasma membrane or the cytoplasm. Recent evidence suggested that several proteins located in the nucleus could also act as viral sensors. In turn, these important elements are becoming the target for most viruses to evade host immune surveillance. In this review, we focus on the recent progress in the study of viral recognition and evasion.

Regulation of chromatin structure and accessibility determines the transcription activities of genes, which endows the host with function-specific patterns of gene expression. Upon viral infection, the innate immune responses provide the first line of defense, allowing rapid production of variegated antiviral cytokines. Knowledge on how chromatin accessibility is regulated during host defense against viral infection remains limited. Our previous work found that the nuclear matrix protein SAFA surveilled viral RNA and regulated antiviral immune genes expression. However, how SAFA regulates the specific induction of antiviral immune genes remains unknown. Here, through integration of RNA-seq, ATAC-seq and ChIP-seq assays, we found that the depletion of SAFA specifically decreased the chromatin accessibility, activation and expression of virus induced genes. And mutation assays suggested that the RNA-binding ability of SAFA was essential for its function in regulating antiviral chromatin accessibility. RIP-seq results showed that SAFA exclusively bound with antiviral related RNAs following viral infection. Further, we combined the CRISPR-Cas13d mediated RNA knockdown system with ATAC-qPCR, and demonstrated that the binding between SAFA and according antiviral RNAs specifically mediated the openness of the corresponding chromatin and following robust transcription of antiviral genes. Moreover, knockdown of these associated RNAs dampened the accessibility of related genes in an extranuclear signaling pathway dependent manner. Interestingly, VSV infection cleaved SAFA protein at the C-terminus which deprived its RNA binding ability for immune evasion. Thus, our results demonstrated that SAFA and the interacting RNA products collaborated and remodeled chromatin accessibility to facilitate antiviral innate immune responses.

Toxoplasma gondii is an important neurotropic pathogen that establishes latent infections in humans that can cause toxoplasmosis in immunocompromised individuals. It replicates inside host cells and has developed several strategies to manipulate host immune responses. However, the cytoplasmic pathogen-sensing pathway that detects T. gondii is not well-characterized. Here, we found that cyclic GMP-AMP synthase (cGAS), a sensor of foreign dsDNA, is required for activation of anti-T. gondii immune signaling in a mouse model. We also found that mice deficient in STING (Stinggt/gt mice) are much more susceptible to T. gondii infection than WT mice. Of note, the induction of inflammatory cytokines, type I IFNs, and interferon-stimulated genes in the spleen from Stinggt/gt mice was significantly impaired. Stinggt/gt mice exhibited more severe symptoms than cGAS-deficient mice after T. gondii infection. Interestingly, we found that the dense granule protein GRA15 from T. gondii is secreted into the host cell cytoplasm and then localizes to the endoplasmic reticulum, mediated by the second transmembrane motif in GRA15, which is essential for activating STING and innate immune responses. Mechanistically, GRA15 promoted STING polyubiquitination at Lys-337 and STING oligomerization in a TRAF protein-dependent manner. Accordingly, GRA15-deficient T. gondii failed to elicit robust innate immune responses compared with WT T. gondii. Conse quently, GRA15-/- T. gondii was more virulent and caused higher mortality of WT mice but not Stinggt/gt mice upon infection. Together, T. gondii infection triggers cGAS/STING signaling, which is enhanced by GRA15 in a STING- and TRAF-dependent manner. [ABSTRACT FROM AUTHOR]

IFN regulatory factor 3 (IRF3) is critical for the transcription of type I IFNs in defensing virus and promoting inflammatory responses. Although several kinds of posttranslational modifications have been identified to modulate the activity of IRF3, whether atypical ubiquitination participates in the function regulation, especially the DNA binding capacity of IRF3, is unknown. In this study, we found that the ovarian tumor domain containing deubiquitinase OTUD1 deubiquitinated IRF3 and attenuated its function. An atypical ubiquitination, K6-linked ubiquitination, was essential for the DNA binding capacity of IRF3 and subsequent induction of target genes. Mechanistically, OTUD1 cleaves the viral infection-induced K6-linked ubiquitination of IRF3, resulting in the disassociation of IRF3 from the promoter region of target genes, without affecting the protein stability, dimerization, and nuclear translocation of IRF3 after a viral infection. Otud12/2 cells as well as Otud12/2 mice produced more type I IFNs and proinflammatory cytokines after viral infection. Otud12/2 mice were more resistant to lethal HSV-1 and VSV infection. Consistent with the former investigations that IRF3 promoted inflammatory responses in LPS-induced sepsis, Otud12/2 mice were more susceptible to LPS stimulation. Taken together, our findings revealed that the DNA binding capacity of IRF3 in the innate immune signaling pathway was modulated by atypical K6-linked ubiquitination and deubiquitination process, which was regulated by the deubiquitinase OTUD1. [ABSTRACT FROM AUTHOR]

Platelet factor 4 (PF4) is an anti-Plasmodium component of platelets. It is expressed in megakaryocytes and released from platelets following infection with Plasmodium. Innate immunity is crucial for the host anti-Plasmodium response, in which type I interferon plays an important role. Whether there is cross-talk between innate immune signaling and the production of anti-Plasmodium defense peptides is unknown. Here we demonstrate that E74, like ETS transcription factor 4 (ELF4), a type I interferon activator, can help protect the host from Plasmodium yoelii infection. Mechanically, ELF4 binds to the promoter of genes of two C-X-C chemokines, Pf4 and pro-platelet basic protein (Ppbp), initiating the transcription of these two genes, thereby enhancing PF4-mediated killing of parasites from infected erythrocytes. Elf4-/- mice are much more susceptible to Plasmodium infection than WT littermates. The expression level of Pf4 and Ppbp in megakaryocytes from Elf4-/- mice is much lower than in those from control animals, resulting in increased parasitemia. In conclusion, our study uncovered a distinct role of ELF4, an innate immune molecule, in host defense against malaria. [ABSTRACT FROM AUTHOR]